Metastatic melanoma is a highly lethal cancer that is associated with the greatest number of productive years of life lost of any solid tumor. Recently significant progress has been made in treating metastatic melanoma by commandeering the immune system through targeting the checkpoint proteins CTLA-4 and PD- 1. Durable response rates of 20-30% in patients with metastatic disease receiving anti-CTLA-4 and anti-PD-1 therapies have generated tremendous promise for a group of individuals whose mean survival had historically been measured in months. However, a significant gap exists in our knowledge of how to improve the effectiveness of checkpoint blockade specifically focused on the question of whether adding a cytotoxic anti- tumor treatment to checkpoint blockade can significantly augment the number of patients who benefit from this form of immunologic treatment. Because systemic chemotherapy treatments are frequently immunosuppressive and have limited effectiveness against melanoma, they are not ideal to utilize in conjunction with immunotherapeutic strategies. Uniquely, melanoma has an array of regional therapeutic strategies that are currently employed for patients with locally advanced disease. The most commonly utilized of these treatments is isolated regional chemotherapy infusions which have complete response rates in the range of 30-50%. Surprisingly, little is know as to whether tumors treated regionally generate an immune response or if these treatments can be synergistic with checkpoint blockade therapy. Therefore, the overall goal of this project is to determine if various regional anti-tumor chemotherapeutic treatments currently utilized in melanoma can augment the immunologic effectiveness of systemic checkpoint blockade therapy and to define the optimal way to deliver this form of combinational therapy. Our group specializes in utilizing preclinical models of advanced extremity melanoma to optimize local disease control and develop unique treatment strategies that are rapidly translated into phase I and II clinical trials. This work has generated our centra hypothesis that regional chemotherapy can generate an immune-activating response which could augment the therapeutic potential of immune checkpoint blockade therapy in overcoming tumor-induced immunosuppression. Integral to this proposal is our use of immunocompetent mice to define how the immune system contributes to local and systemic responses to our optimized regional treatment of melanoma. The scientific rationale behind the proposed specific aims in this grant is to not only determine if regional chemotherapy can render melanoma visible to the immune system but also to allow us to rapidly translate our findings into human trials by defining proper dosing and timing relative to checkpoint blockade. The projects specific aims are: SA 1. Explore the immunologic properties of novel regional chemotherapeutics, determining the optimal dose of these agents to stimulate innate immune activation and generate an adaptive anti-tumor immune response and defining the mediators of this response. SA 2. Determine if targeting immunosuppressive elements of the tumor microenvironment increases the magnitude and duration of anti-tumor immune responses generated by regional chemotherapeutic treatment. SA 3. Establish whether regional therapy combined with adjuvant antibody treatments to minimize immunosuppressive elements in the microenvironment can augment the induction of anti-tumor responses induced by various checkpoint blockade thera-pies and define the optimal timing for this approach. The impact of the studies proposed in this grant will be to either limit further use of regional therapies or define a new role for them in melanoma as part of combinational trials using checkpoint blockade therapy that can be rapidly translated into our current portfolio of clinical trials.